We are studying the effect of beta-6 amino acid on the alpha and beta assembly to produce tetrameric hemoglobin using time resolved spectroscopy. Hb S usually comprises about 40% if the total hemoglobin in the red cells of individuals with sickle cell trait. This lesser amount of Hb S compared to Hb A in persons with sickle cell trait, having a full complement of alpha genes is puzzling. In vitro alpha and beta mixing experiments show that alpha-beta A dimers form more readily than the S dimer. The reasons for this are not readily apparent. The beta-6 amino acid is on the surface of the beta chain and far removed from the alpha-1:beta-1 contact region that is critical for the initial formation of alpha beta dimers. We are now able to systematically test the effects of beta-6 amino acid charge and hydrophobicity on assembly of hemoglobin molecules by engineering various mutations at the beta-6 position. These studies may also be important for understanding reasons for higher levels of the Hb S in sickle cell compared to A type red cells. Furthermore, this difference is an important determinant of the clinical manifestations of sickle cell disease.